Although a potent inhibitor of dihydrofolate reductase, methotrexate (MTX) is effective only against certain forms of cancer, and even these may acquire resistance. Furthermore, since MTX cannot cross the blood brain barrier, treatment of meningeal leukemia and brain tumors necessitate intrathecal or intraventricular injection. However, therapeutic levels of MTX in the brain often result in symptoms of neurotoxicity, the cause of which is unknown. These limitations have stimulated the search for alternative anti-folates. The objective of this proposal is twofold. First, to determine the molecular basis of specificity of pteridines and related compounds so that anti-folates may be designd which are specific for dihydrofolate reductase and which do not interfere with other pteridine utilizing enzymes. Series of substituted pteridines and pyrimidines will be studied as cofactors and inhibitors of the three aromatic amino acid hydroxylases. These enzymes have a specific requirement for a pteridine cofactor, tetrahydrobiopterin (BH4), and are involved in the biosynthesis of serotonin and the catecholamine neurotransmitters. The effects of the pteridines and pyrimidines will also be assessed on dihydropteridine reductase, the enzyme which in vivo regenerates BH4. Any pteridine like drug aimed at dihydrofolate reductase, including pyrimidines as recently demonstrated (Bailey and Ayling, Biochem. Biophys. Res. Comm. 85, 1614-21, 1978), has the potential to restrict BH4 dependent neurotransmitter synthesis. Second, to determine whether the toxicity of MTX, or its metabolites, is caused by inhibition of BH4 biosynthesis, transport, or maintenance. The levels of BH4 and its precursors in rat tissues will be monitored in response to MTX. An understanding of the molecular basis of anti-folate toxicity will enable counteractive measures to be taken. The structure-activity relationships will provide the information necessary to make drugs which selectively inhibit only dihydrofolate reductase.